High-Salt Tolerant Single-Strand DNA Binding Protein
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Solution Overview
Problem
Current single-strand DNA-binding proteins (SSB) are ineffective at high salt concentrations, limiting their ability to form stable nucleoprotein complexes, which is essential for applications like next-generation DNA sequencing and nucleic acid amplification that require high-salt buffers.
Innovation Solution
Development of a single-strand DNA-binding protein (SSB) that maintains at least 50% of its maximum ssDNA binding capability in the presence of 500 mM sodium ions, utilizing the amino acid sequence from Salinibacter ruber or its derivatives, which forms stable octameric or polymeric quaternary structures under high salt conditions, ensuring effective binding and dehybridization of ssDNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional SSB is used, then ssDNA binding is effective at low salt concentrations, but binding capability is lost at high salt concentrations (500 mM sodium ions)
Solution Approach 1:
The invention changes the amino acid sequence parameters of the SSB protein by introducing specific mutations (e.g., replacing residues at positions 17, 71, and 106 with positively charged amino acids like lysine or arginine). These parameter changes in the protein structure enable the SSB to maintain stable electrostatic interactions with ssDNA even in high salt conditions, thereby resolving the contradiction between binding reliability and salt tolerance.
Solution Approach 2:
The invention creates a composite functional protein by combining wild-type SSB sequences with mutated regions that have enhanced salt resistance. This composite structure integrates the high-affinity binding domain of wild-type SSB with the salt-tolerant properties of mutated domains, achieving both reliable ssDNA binding and adaptability to high salt concentrations.
2Productivity
If high salt concentration (500 mM sodium ions) is used in reaction mixture, then nucleic acid amplification and sequencing can be performed, but conventional SSB cannot form stable nucleoprotein complexes
Solution Approach 1:
The mutated SSB protein has altered electrostatic properties due to positively charged amino acid substitutions that enhance its ability to compete with sodium ions for binding to ssDNA. This parameter change in charge distribution allows the nucleoprotein complex to remain stable at 500 mM sodium ion concentrations, enabling productive amplification reactions under high-salt conditions.
3Reliability
If SSB is used to protect ssDNA from nuclease digestion, then DNA integrity is maintained, but protection is ineffective at high salt concentrations
Solution Approach 1:
The amino acid sequence mutations create a more robust protein-DNA interface with enhanced electrostatic attraction. This parameter change in binding strength ensures that the protective nucleoprotein complex remains intact at high salt concentrations where conventional SSB would dissociate, thereby extending the protective function across a broader salt concentration range.
Data Source
AI summary
The present invention relates to the use of a single-strand DNA binding protein (SSB) which exhibits at least 50% of its maximum ssDNA binding capability in the presence of 500 mM of sodium ions, to dehybridize a DNA molecule or to prevent hybridisation of a complementary ssDNA, wherein the SSB comprises the amino acid sequence of SEQ ID NO:1 or an amino acid sequence which is at least 75% identical to SEQ ID NO:1, or a functional fragment thereof, and wherein the DNA molecule or ssDNA is present in or exposed to a solution containing one or more of the following (i) at least 350 mM of sodium ions; (ii) at least 50 mM of potassium ions; (iii) at least 150 mM of magnesium ions; or (iv) at least 200 mM of calcium ions.